Supplementary Information - Nature Research...37005 Ceske Budejovice and Institute of Nanobiology...

1

Supplementary Information

Dynamics and hydration explain failed functional transformation in

dehalogenase design

Jan Sykora1†

, Jan Brezovsky2†

, Tana Koudelakova2†

, Maryna Lahoda3, Andrea Fortova

2,

Tatsiana Chernovets1, Radka Chaloupkova

2, Veronika Stepankova

2, Zbynek Prokop

2,4, Ivana

Kuta Smatanova3, Martin Hof

1*, Jiri Damborsky

2,4*

1 J. Heyrovský Institute of Physical Chemistry ASCR, v. v. i., Dolejškova 3, 182 23 Prague 8,

Czech Republic

2 Loschmidt Laboratories, Department of Experimental Biology and Research Centre for Toxic

Compounds in the Environment RECETOX, Faculty of Science, Masaryk University,

Kamenice 5/A13, 625 00 Brno, Czech Republic

3 Faculty of Science, University of South Bohemia in Ceske Budejovice, Branisovska 31,

37005 Ceske Budejovice and Institute of Nanobiology and Structural Biology ASCR, Zamek

136, 37333 Nove Hrady, Czech Republic

4 International Clinical Research Center, St. Anne's University Hospital Brno, Pekarska 53, 656

91 Brno, Czech Republic

†These authors contributed equally to this work

*To whom correspondence should be addressed. E-mail: [email protected],

[email protected]

Nature Chemical Biology: doi:10.1038/nchembio.1502

2

Table of contents

Supplementary note .................................................................................................................... 3

Molecular modeling .................................................................................................................. 3

Supplementary results ................................................................................................................ 6

Supplementary Figure 1. Circular dichroism spectra of studied enzymes................................ 6

Supplementary Figure 2. Comparison of the residues involved in the transplantation. ........... 7

Supplementary Figure 3. Superposition of the catalytic pentads. ............................................. 8

Supplementary Figure 4. MALDI-TOF MS spectra of DhaA12 and DhaA12-coumarin

complex. .................................................................................................................................... 9

Supplementary Figure 5. Acrylamide quenching of coumarin fluorescence for the haloalkane

dehalogenase variants. ............................................................................................................ 10

Supplementary Figure 6. Structural formula of the fluorescent coumarin probe. .................. 11

Supplementary Figure 7. Two lowest-energy conformations of the coumarin probe. ........... 12

Supplementary Figure 8. Three lowest-energy conformations of the covalent linker. ........... 13

Supplementary Figure 9. Five reactive binding modes of the probe bound in DbjA. ............ 14

Supplementary Figure 10. Three reactive binding modes of the probe bound in DhaA. ....... 15

Supplementary Figure 11. Five reactive binding modes of the probe bound in DhaA12. ..... 16

Supplementary Table 1. Characteristics of enzymes used in this study. ................................ 17

Supplementary Table 2. Enantioselectivity of wild type and variants with bromoalkanes

and bromoester. .................................................................................................................. 18

Supplementary Table 3. Data collection and refinement ........................................................ 19

statistics for the crystal structure of DhaA12. ......................................................................... 19

Supplementary Table 4. Parameters characterizing the overall time-dependent fluorescence

shift. ........................................................................................................................................ 20

Supplementary Table 5. Parameters describing the dynamics and hydration of wild types and

DhaA12 mutant. ...................................................................................................................... 21

Supplementary Table 6. Variable residues in the second and the third shell of the active sites

of DbjA and DhaA12. ............................................................................................................. 22

Supplementary Table 7. Atom types and charges for the fluorescent probe used in the

simulations. ............................................................................................................................. 23

Supplementary Table 8. Atom types and charges for the covalent linker bound to the aspartic

acid residue used in the simulations. ...................................................................................... 24

Supplementary Table 9. Binding energies of individual binding modes of the fluorescent

probe. ...................................................................................................................................... 25

Supplementary references ....................................................................................................... 26


3

Supplementary note

Molecular modeling

Preparation of structures for molecular modeling. Crystal structures of the haloalkane

dehalogenases were obtained from RSCB PDB database using following accession codes:

1CQW (DhaA), 3SK0 (DhaA12) and 3A2M (DbjA). The catalytic histidine was substituted by

phenylalanine in all protein structures and the mutations Val172Ala, Ile209Leu and Gly292Ala

were modeled to the crystal structure of DhaA to achieve correspondence with the

experimentally used haloalkane dehalogenase DhaA from the strain NCIMB13064

(DDBJ/GenBank/EMBL accession no. AF060871). All mutations were designed in

PyMOL 1.4.1 (Schrödinger). Hydrogen atoms were added to the protein structures using H++

server at pH 7.51.

Molecular docking. The fluorescent probe consisting of coumarin 120 dye carrying

covalent alkyl-halogen linker (Supplementary Fig. 6a) was built in PyMOL 1.4.1. The

geometry of probe molecule was optimized using AM1 semi-empirical quantum chemical

method2 implemented in the program MOPAC2009 (Stewart Computational Chemistry). The

structures of the enzymes and the fluorescent probe were prepared for docking calculation by

MGLTools 1.5.03. The fluorescent probe was docked into the active sites of all three studied

enzymes using the program AUTODOCK 3.054. The atomic and electrostatic maps were

calculated using AUTOGRID 3.064. Range of grid maps was set to 95 95 95 grid points

with spacing 0.25 Å centered on CG atom of substituted phenylalanine to cover the whole

active site and the main tunnel of both enzymes. In total, 250 docking calculations were

performed employing the Lamarckian Genetic Algorithm with the following parameters: initial

population size 150, maximum of 1.5 106 energy evaluations and 27,000 generations, elitism

value 1, mutation rate 0.02 and cross-over rate 0.8. The local search was based on the pseudo

Solis and Wets algorithm with a maximum of 300 iterations per local search5. The final

orientations from each docking were clustered with a clustering tolerance for the

root-mean-square positional deviation of 2 Å.

Parameterization of modified amino acid residue for molecular dynamics simulation. The

novel residue representing the fluorescent probe covalently bound to the nucleophile of an

aspartic acid (Supplementary Fig. 6b) was parameterized by analogy with the Cornell et al.

force field6. The only exception was the aromatic oxygen that was present in the moiety of

fluorescent probe. The required parameters were obtained from the VanBeek et al7. The novel

residue was constructed from two fragments (i) the coumarin probe capped with

N-methylamide (NME) residue (Supplementary Fig. 7), and (ii) the covalent linker capped with

acetyl (ACE) residue covalently bound to the aspartic acid residue capped with NME and ACE


4

residues (Supplementary Fig. 8). The coumarin fragment was modeled in two lowest-energy

conformations (Supplementary Fig. 7), while the second fragment was modeled in three

lowest-energy conformations keeping the helical conformation of the aspartic acid residue

(Supplementary Fig. 8). The geometry of all lowest-energy conformations were energy

minimized with MP2/6-31G* wave function using Gaussian09 program revision C.01

(Gaussian, 2009). The partial atomic charges of the novel residue were obtained using RESP

ESP charge Derive 2.0 server8,9

, with HF/6-31G* level of theory. The charges on the fragments

were developed employing RESP-A1A charge model, using multi-conformation

multi-orientation RESP fits. During the fitting procedure, the charges on the capping NME and

ACE residues were constrained to zero. The charges on the four peptide bond atoms

(N43, H44, C45 and O46) of the second fragment were constrained to the values corresponding

to the charges on respective atoms of the electro-neutral residues from the Cornell et al. force

field6. The charges and atom types for both fragments are provided in the Supplementary

Table 7 and Supplementary Table 8.

Molecular dynamics simulation. The covalently bound alkyl-enzyme intermediates were

manually modeled from the enzyme-substrate complexes obtained from the docking

calculations. All enzyme-substrate complexes complying with the basic geometrical

prerequisites of SN2 reaction in the haloalkane dehalogenases10

were selected (Supplementary

Fig. 9-11), and the alkyl-enzyme intermediates were prepared by deleting the chloride atom and

placing the carbon atom 1 Å closer to the nucleophile oxygen atom of the aspartic acid residue

106 (DhaA and DhaA12) and 103 (DbjA). Water molecules from the crystal structure were

added to the systems to their original positions with the exception of water molecules

overlapping with the docked probes. The systems were neutralized by adding 16, 21 and 7 Na+

ions to DhaA, DhaA12 and DbjA complexes, respectively, using Tleap module of

AmberTools11 (University of California, 2010). Using the same module, an octahedron of

TIP3P water molecules11

was added to the distance of 10 Å from any solute atom in the

systems. Energy minimization and MD simulations were carried out in PMEMD module of

AMBER11 (University of California, 2010) using ff99SB force field12

. The investigated

systems were minimized by 500 steps of steepest descent followed by 500 steps of conjugate

gradient in five rounds of decreasing harmonic restraints. The restraints were applied as

follows: 500 kcal.mol-1

.Å-2

on all heavy atoms of protein, and then 500, 125, 25 and

0 kcal.mol-1

.Å-2

on backbone atoms only. The consequent MD simulations employed periodic

boundary conditions, the particle mesh Ewald method for treatment of the electrostatic

interactions13,14

, 10 Å cutoff for nonbonded interactions, and 2 ft time step with the SHAKE

algorithm to fix all bonds containing hydrogens15

. Equilibration simulations consisted of two

steps: (i) 20 ps of gradual heating from 0 to 300 K under constant volume, using a Langevin


5

thermostat with collision frequency of 1.0 ps-1

, and with harmonic restraints of

5.0 kcal.mol-1

.Å-2

on the position of all protein atoms, and (ii) 2000 ps of unrestrained MD at

300 K using the Langevin thermostat, and constant pressure of 1.0 bar using pressure coupling

constant of 1.0 ps. Finally, production MD simulations were run for 50 ns with the same

settings as the second step of equilibration MD. Coordinates were saved in 5 ps interval, and

the trajectories were analyzed using Ptraj and Cpptraj modules of AmberTools11, and

visualized in Pymol 1.4.1 and VMD 1.8.916

. The average binding energies of each binding

mode were evaluated using Molecular-Mechanics/Generalized-Born Surface Area17,18

. Every

second frame from the equilibrated part of the molecular dynamics trajectories was selected for

the analysis. The following settings were used for the calculation: PBradii were set to mbondi2

and Generalized-Born model to 518

. The analysis was performed with MMPBSA.py python

script of AmberTools11. The binding modes with the lowest energies were then selected for the

detailed analysis of each system (Supplementary Table 9).


6

Supplementary results

Supplementary Figure 1. Circular dichroism spectra of studied enzymes. All enzymes except

DhaA07 and DhaA09 exhibited circular dichroism spectra typical of folded /-proteins with

a predominately -helical content. Collected circular dichroism data are expressed in terms of

the mean residue ellipticity (ΘMRE).


7

a b

Supplementary Figure 2. Comparison of the residues involved in the transplantation.

(a) Active site and tunnel lining residues variable between DbjA (red) and DhaA (magenta).

(b) Residues transplanted from DbjA (red) to DhaA12 (blue). EBR fragment is show as ribbon.


8

Supplementary Figure 3. Superposition of the catalytic pentads. Pentads of DhaA12 (in blue)

and DbjA (in red) are represented by sticks. The 2Fo-Fc electron density map of the DhaA12

pentad contoured at 1σ represented by mesh.


9

Supplementary Figure 4. MALDI-TOF MS spectra of DhaA12 and DhaA12-coumarin

complex. Black spectrum of the free enzyme (peak 1) is compared with red spectrum of the

enzyme-probe complex (peak 2). Seven consecutive mass spectra were recorded for each

sample and average mass values were calculated (RSD ≤ 0.1%). Difference of average masses

of enzymes and enzyme-probe complex corresponds to the mass of the coumarin probe. The

proteins contain the point mutation in the catalytic histidine His283Phe.


10

Supplementary Figure 5. Acrylamide quenching of coumarin fluorescence for the haloalkane

dehalogenase variants. Stern-Volmer plots for DbjA (■), DhaA (Δ) and DhaA12 (○) provided

Stern-Volmer constants of 0.29 ± 0.04 M-1

, 0.11 ± 0.02 M-1

and 0.23 ± 0.06 M-1

respectively.

The linearity of those graphs indicates that those enzymes are specifically labeled. The

accessibility of the coumarin dye to the acrylamide molecules is substantially higher in the case

of the mutants DbjA and DhaA12 than in the case of DhaA. Stern-Volmer constant for DbjA

and DhaA12 are comparable, confirming the fact that the tunnel architecture of these enzymes

is similar. The emission spectra recorded before and after the quenching procedure did not

show any differences for all herein investigated mutants further indicating that the coumarin

dye possesses single location within the protein scaffold. Three experiments were performed

for DhaA and DbjA, and duplicate measurements were carried out in the case of DhaA12. Data

represent mean values ± s.d.


11

a

b

Supplementary Figure 6. Structural formula of the fluorescent coumarin probe. Unbound

form (a) and its alkyl-intermediate complex with aspartic acid (b).


12

Supplementary Figure 7. Two lowest-energy conformations of the coumarin probe. Probe is

capped with NME residue (atoms 27-32) employed for the fragment parameterization. The

atoms are numbered in concert with Supplementary Table 7.


13

Supplementary Figure 8. Three lowest-energy conformations of the covalent linker. Linker is

capped with ACE residue (atoms 59-64) covalently bound to the aspartic acid residue capped

with NME (atoms numbered 47-52) and ACE (atoms 53-58) residues employed for the

fragment parameterization. The atoms are numbered in concert with Supplementary Table 8.


14

Supplementary Figure 9. Five reactive binding modes of the probe bound in DbjA. Probe is

visualized with yellow sticks and enzyme as grey cartoon. The catalytic nucleophile and the

two halide-stabilizing residues are in cyan sticks.


15

Supplementary Figure 10. Three reactive binding modes of the probe bound in DhaA. Probe

is visualized with yellow sticks and enzyme as grey cartoon. The catalytic nucleophile and the



16

Supplementary Figure 11. Five reactive binding modes of the probe bound in DhaA12. Probe

is visualized with yellow sticks and enzyme as grey cartoon. The catalytic nucleophile and the



17

Supplementary Table 1. Characteristics of enzymes used in this study.

Enzyme Mutations Engineered region Tm (C)a Reference

DhaA None None 50.5 0.3 This study

DhaA

+H272F H272F Catalytic base NT

b

19

DhaA07 ERB fragment + W141F + P153A ERB fragment unfolded This study

DhaA08 ERB fragment + W141F + P153A +

F155A ERB fragment 35.0 0.8 This study


F155A + V256A Main access tunnel unfolded This study


F155A + V256A + A183V Main access tunnel 40.5 0.6 This study


F155A + V256A + A183V + C187G Main access tunnel 42.5 0.8 This study

DhaA12

ERB fragment + W141F + P153A +

F155A + V256A + A183V + C187G

+ G182R + K186G

Main access tunnel 44.6 0.2 This study

DhaA12

+H283F

ERB fragment + W141F + P153A +

F155A + V256A + A183V + C187G

+ G182R + K186G + H283F

Catalytic base NTb This study

DbjA None None 53.6 0.6 20,21

DbjA

+H280F H280F Catalytic base NT

b

19

ERB fragment relates to the insertion of His-His-Thr-Glu-Val-Ala-Glu-Glu-Gln-Asp-His between the residues 141

and 142 of DhaA wild type. Cumulatively added mutations are in bold. The point mutation in the catalytic base

(His272Phe, His283Phe and His280Phe) enables covalent binding of fluorescence probe required for

time-dependent fluorescence experiments. aTm – melting temperature; data represent mean values ± s.d. calculated

from triplicate experiments bNT – not tested


18

Supplementary Table 2. Enantioselectivity of wild type and variants with bromoalkanes

and bromoester.

Enzyme E-value

2-Bromopentane 2-Bromohexane 2-Bromoheptane Ethyl 2-bromopropionate

DhaA 8 4 2 72

DhaA08 5 4 2 163

DhaA10 9 11 2 > 200

DhaA11 8 12 2 > 200

DhaA12 7 11 10 > 200

DbjAa 145 68 28 > 200

Data was fitted by nonlinear regression based on the simulation with the standard error of the fit

between 5 and 25%. a data from

20.


19

Supplementary Table 3. Data collection and refinement

statistics for the crystal structure of DhaA12.

DhaA12

Data collection

Space group P212121

Cell dimensions

a, b, c (Å) 51.58, 68.69, 84.37

() 90.00, 90.00, 90.00

Resolution (Å) 31.39–1.78 (1.88-1.78)a

Rmerge 8.4 (37)

I / I 14.4 (4.2)

Completeness (%) 100.0 (99.9)

Redundancy 4.1 (4.1)

Refinement

Resolution (Å) 1.78

No. reflections 34,658

Rwork / Rfree 0.176/0.207

No. atoms

Protein 2,537

Chloride ion 1

Water 270

Non-H atoms 2,808

B-factors (Å2)

Protein 16.7

Ligand/ion 12.7

Water 24.5

R.m.s. deviations

Bond lengths (Å) 0.007

Bond angles () 0.024

Parameters were obtained from a single crystal of DhaA12 protein.

a The highest-resolution shell is shown in parentheses.


20

Supplementary Table 4. Parameters characterizing the overall time-dependent fluorescence

shift.

Enzyme 0

(cm-1

)

(cm

-1)

1 (ns)

(A1)

2 (ns)

(A2)

3 (ns)

(A3) r (ns) % obs.

DhaA 23300±60 950±80 0.2

(310)

1.3

(340)

12.6

(200) 4.1±0.3 90±5

DhaA12 23300±70 1000±100 0.2

(400) -

9.1

(400) 3.8±0.4 85±6

DbjA 23400±60 1300±140 < 0.1

(280)

1.4

(220)

11.0

(370) 2.8±0.3 62±9

0 corresponds to the emission maximum of “time zero spectrum” which is a spectrum emitted from the

completely non-relaxed Franck-Condon state and which was determined by the modified “time 0

estimation” as described previously19

. The rest of the given parameters was obtained by the analysis of the

time dependence of time resolved emission spectra (TRES): ) stands for the emission maximum of the

spectrum from fully relaxed state which was obtained by extrapolation of (t) to t . represents the

overall dynamic Stokes Shift derived as 0 - ultiexponential fitting of time evolution of the

TRES peak maximum (t), and analysis of the autocorrelation curve C(t): i are the fitted relaxation times,

Ai correspond to the amplitudes, r is the integral relaxation time, and % obs. corresponds to the percentage

of the solvent relaxation process captured with the given experimental time resolution of 30 ps. Four

experiments were performed for DhaA and DbjA, and duplicate measurements were carried out in the case

of DhaA12. Data represent mean values ± s.d. i and Ai parameters were gained by the non-linear least

square fitting with 3-exponential function. Fitting procedure yielded standard deviation lower than 10% for

all the parameters.


21

Supplementary Table 5. Parameters describing the dynamics and hydration of wild types

and DhaA12 mutant.

a data from time-dependent fluorescence shift experiments; data represent mean values ± s.d. calculated from

four experiments for DhaA and DbjA, and two measurements for DhaA12. b data from molecular dynamics

simulations; data represent mean values ± s.d. calculated from 3,000 snapshots.

Enzyme

Dynamics Hydration

Integral relaxation

time [ns]a

Integral B-factor of the

tunnel mouth near dye [Å2]

b

Dynamic Stokes

shift [cm-1

]a

Average number of water

molecules around dyeb

DhaA 4.1± 0.3 132 950±80 6±2

DhaA12 3.8± 0.4 217 1000±100 6±2

DbjA 2.8± 0.3 315 1300±140 10±2


22

Supplementary Table 6. Variable residues in the second and the third shell of

the active sites of DbjA and DhaA12.

Residues Location

DbjA DhaA12

Phe66 Met69 Second shell

Gln102 His105 Second shell

Thr106 Ser109 Second shell

Met132 Ile135 Second shell

Ile185 Val188 Second shell

Val210 Leu213 Second shell

Leu230 Val235 Second shell

Val255 Ile258 Second shell

Ile44 Leu47 Third shell

Ala101 Ile104 Third shell

Ala109 Gly112 Third shell

Phe125 Cys128 Third shell

Ala233 Tyr236 Third shell

His234 Met237 Third shell

Ser256 Pro259 Third shell

Leu275 Ile278 Third shell


23

Supplementary Table 7. Atom types and charges for the fluorescent probe used in the

simulations.

Atom number Atom type Charge

1 N2 -0.8691

2 H 0.3788

3 H 0.3788

4 CA 0.3555

5 CA -0.2906

6 HA 0.1770

7 CA -0.1881

8 HA 0.1741

9 CA 0.0162

10 CA 0.1939

11 CA -0.2725

12 HA 0.1773

13 OA -0.3635

14 CA 0.7418

15 O -0.5652

16 CA 0.0595

17 CT -0.1794

18 HC 0.0799

19 HC 0.0799

20 HC 0.0799

21 CA -0.1896

22 CT -0.0634

23 HC 0.0502

24 HC 0.0502

25 C 0.5745

26 O -0.5863

Atom numbers are defined in Supplementary Fig. 7. Atom types accordingly to Cornell et al. force field6.


24

Supplementary Table 8. Atom types and charges for the covalent linker bound to the aspartic

acid residue used in the simulations.

Atom number Atom type Charge

1 N -0.4520

2 H 0.2592

3 CT 0.0703

4 H1 0.0632

5 H1 0.0632

6 CT 0.1685

7 H1 0.0333

8 H1 0.0333

9 OS -0.4819

10 CT 0.1700

11 H1 0.0315

12 H1 0.0315

13 CT 0.1970

14 H1 0.0266

15 H1 0.0266

16 OS -0.4681

17 CT 0.1421

18 H1 0.0196

19 H1 0.0196

20 CT 0.0397

21 HC 0.0104

22 HC 0.0104

23 CT 0.0190

24 HC -0.0126

25 HC -0.0126

26 CT 0.0131

27 HC -0.0046

28 HC -0.0046

29 CT 0.0142

30 HC 0.0252

31 HC 0.0252

32 CT 0.0353

33 H1 0.0768

34 H1 0.0768

35 OS -0.3987

36 C 0.7481

37 O -0.5573

38 CT -0.2840

39 HC 0.0969

40 HC 0.0969

41 CT 0.1284

42 H1 0.0192

43 N -0.4156

44 H 0.2718

45 C 0.5972

46 O -0.5679

Atom numbers are defined in Supplementary Fig. 8. Atom types accordingly to Cornell et al. force field6.


25

Supplementary Table 9. Binding energies of individual binding modes of the fluorescent

probe.

Enzyme Binding mode

Enzyme-probe binding energy [kcal/mol]

DbjA DbjA_18 DbjA_21 DbjA_28 DbjA_30 DbjA_38

-13±3 -11±2 -10±2 -19±2 -11±2

DhaA DhaA_4 DhaA_10 DhaA_33

- - -16±3 -21±2 -11±2

DhaA12 DhaA12_4 DhaA12_28 DhaA12_40 DhaA12_45 DhaA12_65

-23±3 -9±3 -9±3 -11±2 -8±1

Individual binding modes are depicted in Supplementary Fig. 9-11. Binding modes selected for further analysis are

in bold. Data represent mean values ± s.d. calculated from 3,000 snapshots.


26

Supplementary references

1. Gordon, J. C. et al. Nucleic Acids Res. 33, W368–371 (2005).

2. Dewar, M. J. S., Zoebisch, E. G., Healy, E. F. & Stewart, J. J. P. J. Am. Chem. Soc. 107,

3902–3909 (1985).

3. Sanner, M. F. J. Mol. Graph. Model. 17, 57–61 (1999).

4. Morris, G. M. et al. J. Comput. Chem. 19, 1639–1662 (1998).

5. Solis, F. J. & Wets, R. J. B. Math. Oper. Res. 6, 19–30 (1981).

6. Cornell, W. D. et al. J. Am. Chem. Soc. 117, 5179–5197 (1995).

7. VanBeek, D. B., Zwier, M. C., Shorb, J. M. & Krueger, B. P. Biophys. J. 92, 4168–4178

(2007).

8. Dupradeau, F.-Y. et al. Phys. Chem. Chem. Phys. 12, 7821–7839 (2010).

9. Vanquelef, E. et al. Nucleic Acids Res. 39, W511–517 (2011).

10. Hur, S., Kahn, K. & Bruice, T. C. Proc. Natl. Acad. Sci. U. S. A. 100, 2215–2219 (2003).

11. Jorgensen, W. L., Chandrasekhar, J., Madura, J. D., Impey, R. W. & Klein, M. L. J. Chem.

Phys. 79, 926–935 (1983).

12. Hornak, V. et al. Proteins 65, 712–25 (2006).

13. Darden, T., York, D. & Pedersen, L. J. Chem. Phys. 98, 10089–10092 (1993).

14. Essmann, U. et al. J. Chem. Phys. 103, 8577–8593 (1995).

15. Ryckaert, J.-P., Ciccotti, G. & Berendsen, H. J. C. J. Comput. Phys. 23, 327–341 (1977).

16. Humphrey, W., Dalke, A. & Schulten, K. J. Mol. Graph. 14, 33–8, 27–8 (1996).

17. Still, C., Tempczyk, A., Hawley, R. & Hendrickson, T. J. Am. Chem. Soc. 112, 6127–6129

(1990).

18. Onufriev, A., Bashford, D. & Case, D. A. Proteins 55, 383–394 (2004).

19. Jesenská, A. et al. J. Am. Chem. Soc. 131, 494–501 (2009).

20. Prokop, Z. et al. Angew. Chem. Int. Ed Engl. 49, 6111–6115 (2010).

21. Chaloupkova, R., Prokop, Z., Sato, Y., Nagata, Y. & Damborsky, J. FEBS J. 278, 2728–

2738 (2011).


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